Guanase is an important enzyme which catalyzes the conversion of guanine to xanthine in the nucleotide metabollism of living bodies. As shown by the following formulae, in the higher animals, the xanthine formed by this enzyme is oxidized to uric acid, but in the lower animals, said uric acid is further decomposed to allantoin and thereafter exhausted as urine. ##STR1##
As the progress of the diagnostic medicine continues, the pathological inspection of the body fluids such as serium has now become important. Thus, in the diagnosis of liver diseases, there are several inspections for estimating the activity of enzymes, such as transaminase (COT,GPT), alkaliphosphatase and gamma-glutamyl-transpeptidase (gamma-GTP) which have been broadly carried out. Among these tests, transaminase and alkaliphosphatase seem not to be good parameters for the liver diseases since they are broadly distributed in the body tissues other than the liver. On the other side, the latter gamma-GTP is valuable for diagnosis of the liver diseases since it is known to be peculiar for the liver diseases (Szasz, G., Rosenthal, P. and Fritzsche, W.; Dt. Med. Wschr., 94, pp. 1911-1917(1969)). Although this gamma-GTP has become an important parameter for the diagnosis of chronic hepatitis, cirrhosis and obstructive jaundice since it keenly reflects to biliary injuries, it is almost inactive to an acute hepatitis. (The titer of this enzyme is almost unchanged by the latter disease.) (Fujisawa, K.; Gan TO Kagakuryoho (Cancer and Chemotherapy) 5, suppl. II, 373-379(1978)). Thus, the assay of this enzyme is not available for the diagnosis of acute hepatitis.
Contrary to the former, it has been known that the titer of guanase is particularly raisen in case of the acute viral hepatitis but is almost not unchanged in case of/chronic hepatitis and cirrhosis (Whitehouse, J. L., Knights, E. M., Santoz, C. L. and Hue, A. C.,: Clin, Chem., 10, p.632(1964); Bel, A., et al.: Presse. Med., 78, pp. 495-499(1970)). Therefore, the assay of the titer of this enzyme is available for the diagnosis of acute viral hepatitis as a reliable tool.
Moreover, the hepatitis followed by blood transfusion which has become a serious social problem is almost caused by non-A and non-B viruses. However, any effective screening method for the protection of these kinds of hepatitis has not been discovered. Recently, it however has become clear that such hepatitis can considerably prevented, if only the blood for transfusion can be preliminarily screened as to the titer of guanase. Thus, the assay of guanase is also useful for preventive medicine.
In turn, according to the increase in the diagnostic value of this enzyme, there are increasing needs for automatic assay of it. However, because of lower titer of this enzyme in normal human blood serum and considerable long time needed for assay, there has heretofore not been existing any appropriate automatic means for assaying this enzyme. Namely, in the present clinical measurement, highly automated analysers are positively introduced. According to such analyser, various kinds of assays are paralelly carried out so as to complete whole assays within 10-15 minutes per each sample. Thus, it is practically impossible to prolong the assaying time only for guanase. Alternatively, even if such prolongation is possible, practice of such alteration makes the introduction of expensive automatic analyser meaningless. In this connection, the assay of guanase has not become common for routine clinical assay.
The object of the present invention is to surprisingly shorten the necessary time for assaying guanase without lowering accuracy by improving known methods used heretofore for assaying this enzyme and to make it possible to introduce said method to an automatic analyzer.
In turn, known representative assaying methods for guanase are enumerated as follows:
(1) PA0 (2) PA0 (3) PA0 (4) PA0 (5) PA0 (I) Guanase reaction step (xanthin-formation step): In this step, the sample solution is added into a buffer solution of substrate (guanine) (pH 7-9) followed during incubation for 10-30 minutes by which the substrate is decomposed to xanthine and ammonia by the action of the guanase in the sample. PA0 (II) Hydrogen peroxide-formation step: In this step, a buffer solution of xanthine oxidase (pH 7-9) is added to the reaction solution of (I) and the xanthine formed by the foregoing step (I) is decomposed to uric acid and hydrogen peroxide. PA0 (III) Coloring reaction step: To the reaction solution of (II), there are simultaneously added MBTH, an aniline derivative and peroxidase so as to form an indamine dye. The foregoing steps can be illustrated by the following scheme: ##STR2##
Roush-Norris Method (Roush, A. and Norris, E. R.; Arch. Biochem, 29, pp.124-129(1950)): This method is based on the difference in the optical absorption between guanine and xanthine. The decreasing rate of guanine is assayed. PA1 Hue-Free Method (Hue, A. C. and Free, A. H.; Clin. Chem. 11. pp.708-715(1965)) is an improvement of above-mentioned method, wherein the measurement is carried out in a borate buffer in order to remove the influence of xanthine oxydase in the sample. (Absorption strength (Molar absorption coefficient) (.epsilon.)=0.535.times.10.sup.4.) PA1 Kalcker method (Kalcker, H. M.; J.Biol. Chem., 167, pp. 429-444, 445-459): In this method, xanthine produced from guanine is furthermore oxidized to uric acid by milk xanthine oxidase. The amount of the uric acid formed is measured by the change of the absorption at 290 nm. .epsilon.=1.23.times.10.sup.4 PA1 Ammonia-assay method PA1 (i) Ellis-Goldberg Method (Ellis, Graham and Goldberg, David M.; Biochem. Med., 6, pp. 380-391(1972)): In this method, the amount of ammonia by-produced when xanthine is formed from guanine is estimated. The assay of ammonia is carried out by reacting alpha-ketoglutaric acid and NADH (reduced type of nicotinic acid amide adeninedinucleotide) with producing ammonia under the exsistence of glutamate dehydrogenase and estimating the amount of NADH consumed. .epsilon.=0.622.times.10.sup.4 PA1 (ii) Ito et al. Method (Susumu Ito, Masahiro Kagawa, Tutomu Kanbara and Takeshi Murakami; Rinshobyori (Clinical Pathology); 23, pp.733-736(1975)): In this method, the amount of the ammonia formed is assayed by utilizing indophenol reaction. .epsilon.=1.96.times.10.sup.4 PA1 Hydrogen peroxide-assay Method PA1 In this method, the amount of the hydrogen peroxide by-produced by the practice of above Kalcker method is measured. PA1 (i) Fritz et al. Method (Fritz, Heintz, Reckel, Sylvia and Kalden, Joachim R.; Enzyme, 24 pp.247-254 (1979)): In this method, the hydrogen peroxide is subjected to the action of catalase under the presence of ethanol to form acetaldehyde, which is then reacted with NAD (Oxidized type of nicotinic acid amide adeninedinucleotide) and acetaldehyde dehydrogenase to form NADH. The change in the amount of the NADH is determined for the assay. .epsilon.=0.624.times.10.sup.4 PA1 (ii) Sugiura et al. Method (Kenji Kato, Tetsuo Adachi, Yoshimasa Ito, Kazuyuki Hirano and Mamoru Sugiura; Lecture Abstract of Sympodium for Analitical Chemistry of Ingredients in the Living Bodies, Japan Pharmaceutical Society Press, pp. 41-44 (1979)): In this method, hydrogen peroxide is reacted with 3-methyl-2-benzothiazolinonehydazone (MBTH), dimethylaniline (DMA) and peroxidase to form an indamine pigment which is then assayed according to a colorimetry. .epsilon.=3.37.times.10.sup.4 PA1 Radioisotope Method (Van Bennekom, C. A., Van Laarhoven, J. P., De Bruyn, C. H. M. M. and Oei, T. L.; J. Clin. Biochem., 16, pp.245-248(1978)):
This method uses a labeled guanine with .sup.14 C and xanthine formed is separated by an electrophoresis, and then the radioactivity of the latter is measured by a scintilation counter. This method has high accuracy but it also requires special equipment and expertise since it uses dangerous .sup.14 C. Therefore, this is hardly available for common clinical test. As hereinbefore described there are many methods for the estimation of guanase activity, however, normally the guanase activity in human blood serum is usually 1-2 IU/1 (1 IU corresponds to the activity of decomposing 1 .mu.mol of substrate per minute at 37.degree. C.) so that the value should be over 2.times.10.sup.4 in order to complete the reaction within 15 minutes. Among all methods mentioned, only Sugiura method of (4)(ii), above is satisfactory to this condition. However, this method has a essential defect that it can not exclude the effect of catalase in serum and therefore it can not reflect on true titer of guanase. Namely, since sodium azide added as the catalase inhibitor does not completely inhibit the catalase activity, if hydrogen peroxide is co-existed with catalase during reaction time, the data will naturally become incorrect. Moreover, the guanase reaction, wherein the substrate is decomposed, is carried out at pH 6.2 in order to stabilize hydrogen peroxide.
The optimal pH of guanase however is approximately at pH 8.0 so that the enzyme exerts about 60% activity as compared with that of the optimal one. This fact causes the prolongation in the reaction time.